Environmental regulation of male fertility is mediated through Arabidopsis transcription factors bHLH89, 91, and 10.

Formation of functional pollen and successful fertilization rely on the spatial and temporal regulation of anther and pollen development. This process responds to environmental cues to maintain optimal fertility despite climatic changes. Arabidopsis transcription factors basic helix-loop-helix (bHLH) 10, 89, and 91 were previously thought to be functionally redundant in their control of male reproductive development, however here we show that they play distinct roles in the integration of light signals to maintain pollen development under different environmental conditions. Combinations of the double and triple bHLH10,89,91 mutants were analysed under normal (200 µmol m-2 s-1) and low (50 µmol m-2 s-1) light conditions to determine the impact on fertility. Transcriptomic analysis of a new conditionally sterile bhlh89,91 double mutant shows differential regulation of genes related to sexual reproduction, hormone signal transduction, and lipid storage and metabolism under low light. Here we have shown that bHLH89 and bHLH91 play a role in regulating fertility in response to light, suggesting that they function in mitigating environmental variation to ensure fertility is maintained under environmental stress.

Barley TAPETAL DEVELOPMENT and FUNCTION1 (HvTDF1) gene reveals conserved and unique roles in controlling anther tapetum development in dicot and monocot plants.

The anther tapetum helps control microspore release and essential components for pollen wall formation. TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is an essential R2R3 MYB tapetum transcription factor in Arabidopsis thaliana; however, little is known about pollen development in the temperate monocot barley. Here, we characterize the barley (Hordeum vulgare L.) TDF1 ortholog using reverse genetics and transcriptomics. Spatial/temporal expression analysis indicates HvTDF1 has tapetum-specific expression during anther stage 7/8. Homozygous barley hvtdf1 mutants exhibit male sterility with retarded tapetum development, delayed tapetum endomitosis and cell wall degeneration, resulting in enlarged, vacuolated tapetum surrounding collapsing microspores. Transient protein expression and dual-luciferase assays show TDF1 is a nuclear-localized, transcription activator, that directly activates osmotin proteins. Comparison of hvtdf1 transcriptome data revealed several pathways were delayed, endorsing the observed retarded anther morphology. Arabidopsis tdf1 mutant fertility was recovered by HvTDF1, supporting a conserved role for TDF1 in monocots and dicots. This indicates that tapetum development shares similarity between monocot and dicots; however, barley HvTDF1 appears to uniquely act as a modifier to activate tapetum gene expression pathways, which are subsequently also induced by other factors. Therefore, the absence of HvTDF1 results in delayed developmental progression rather than pathway failure, although inevitably still results in pollen degeneration.

Chlorophyll fluorescence-based high-throughput phenotyping facilitates the genetic dissection of photosynthetic heat tolerance in African (Oryza glaberrima) and Asian (Oryza sativa) rice.

Rising temperatures and extreme heat events threaten rice production. Half of the global population relies on rice for basic nutrition, and therefore developing heat-tolerant rice is essential. During vegetative development, reduced photosynthetic rates can limit growth and the capacity to store soluble carbohydrates. The photosystem II (PSII) complex is a particularly heat-labile component of photosynthesis. We have developed a high-throughput chlorophyll fluorescence-based screen for photosynthetic heat tolerance capable of screening hundreds of plants daily. Through measuring the response of maximum PSII efficiency to increasing temperature, this platform generates data for modelling the PSII-temperature relationship in large populations in a small amount of time. Coefficients from these models (photosynthetic heat tolerance traits) demonstrated high heritabilities across African (Oryza glaberrima) and Asian (Oryza sativa, Bengal Assam Aus Panel) rice diversity sets, highlighting valuable genetic variation accessible for breeding. Genome-wide association studies were performed across both species for these traits, representing the first documented attempt to characterize the genetic basis of photosynthetic heat tolerance in any species to date. A total of 133 candidate genes were highlighted. These were significantly enriched with genes whose predicted roles suggested influence on PSII activity and the response to stress. We discuss the most promising candidates for improving photosynthetic heat tolerance in rice.

Understanding the support needs of parents of children with obsessive-compulsive disorder: a qualitative descriptive study in the UK.

INTRODUCTION: Caring for a child with obsessive-compulsive disorder (OCD) can be extremely difficult, yet evidence-based support strategies for parents/carers are limited. A detailed understanding of parent support needs is an important first step in intervention development and qualitative research with this focus is currently lacking. In this study, the viewpoints of parents and professionals were used to understand support needs and preferences when caring for a child with OCD. This qualitative descriptive study formed part of a wider UK-based project aimed at developing better support for parents of children with OCD. METHOD: Individual semi-structured interviews (and an optional one-week journal) with a purposive sample of parents of children and young people (CYP) with OCD, aged 8-18, and focus groups (or individual interviews where preferred) with a purposive sample of professionals supporting CYP with OCD. Data comprised transcripts of audio-recorded interviews and focus groups, and text from journals. Analysis was informed by the Framework approach involving inductive and deductive coding, supported by NVivo 12.0 software. Co-production methods were adopted throughout the research process, including the involvement of a parent co-researcher and charity collaborators. RESULTS: Interviews were undertaken with 20 parents, of which 16 completed a journal. Twenty-five professionals took part in a focus group or interview. Five key themes relating to parent support challenges and support needs/preferences were identified (1) Coping with the impact of OCD; (2) Getting help for my child; (3) Understanding parents' role; (4) Making sense of OCD; (5) Joined-up care. CONCLUSION: Parents caring for children with OCD have clear caregiver support needs which are currently not being met. Through triangulation of parent and professional accounts, this study has identified parent support challenges (e.g., emotional impact of OCD, visibility of caring role, misunderstanding about OCD) and support needs/ preferences (e.g., headspace/respite, compassion/sensitivity, guidance on accommodation) to lay the vital foundations for the development of effective parent support interventions. There is now an urgent need to develop and test an intervention to support parents in their caregiving role, with the aim of preventing and/or reducing their levels of burden and distress and ultimately, improving their quality of life.

Rice HEAT SHOCK PROTEIN60-3B maintains male fertility under high temperature by starch granule biogenesis.

Heat stress has a deleterious effect on male fertility in rice (Oryza sativa), but mechanisms to protect against heat stress in rice male gametophytes are poorly understood. Here, we have isolated and characterized a heat-sensitive male-sterile rice mutant, heat shock protein60-3b (oshsp60-3b), that shows normal fertility at optimal temperatures but decreasing fertility as temperatures increase. High temperatures interfered with pollen starch granule formation and reactive oxygen species (ROS) scavenging in oshsp60-3b anthers, leading to cell death and pollen abortion. In line with the mutant phenotypes, OsHSP60-3B was rapidly upregulated in response to heat shock and its protein products were localized to the plastid. Critically, overexpression of OsHSP60-3B enhanced the heat tolerance of pollen in transgenic plants. We demonstrated that OsHSP60-3B interacted with FLOURY ENDOSPERM6(FLO6) in plastids, a key component involved in the starch granule formation in the rice pollen. Western blot results showed that FLO6 level was substantially decreased in oshsp60-3b anthers at high temperature, indicating that OsHSP60-3B is required to stabilize FLO6 when temperatures exceed optimal conditions. We suggest that in response to high temperature, OsHSP60-3B interacts with FLO6 to regulate starch granule biogenesis in rice pollen and attenuates ROS levels in anthers to ensure normal male gametophyte development in rice.

Cytokinin signaling regulates two-stage inflorescence arrest in Arabidopsis.

To maximize reproductive success, flowering plants must correctly time entry and exit from the reproductive phase. While much is known about mechanisms that regulate initiation of flowering, end-of-flowering remains largely uncharacterized. End-of-flowering in Arabidopsis (Arabidopsis thaliana) consists of quasi-synchronous arrest of inflorescences, but it is unclear how arrest is correctly timed with respect to environmental stimuli and reproductive success. Here, we showed that Arabidopsis inflorescence arrest is a complex developmental phenomenon, which includes the arrest of the inflorescence meristem (IM), coupled with a separable "floral arrest" of all unopened floral primordia; these events occur well before visible inflorescence arrest. We showed that global inflorescence removal delays both IM and floral arrest, but that local fruit removal only delays floral arrest, emphasizing their separability. We tested whether cytokinin regulates inflorescence arrest, and found that cytokinin signaling dynamics mirror IM activity, while cytokinin treatment can delay both IM and floral arrest. We further showed that gain-of-function cytokinin receptor mutants can delay IM and floral arrest; conversely, loss-of-function mutants prevented the extension of flowering in response to inflorescence removal. Collectively, our data suggest that the dilution of cytokinin among an increasing number of sink organs leads to end-of-flowering in Arabidopsis by triggering IM and floral arrest.

Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

Heat stress responses vary during floret development in European spring barley cultivars.

The Poaceae, or grasses, include many agriculturally important cereal crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Barley is a widely grown cereal crop used for stock feed, malting and brewing. Abiotic stresses, particularly global warming, are the major causes of crop yield losses by affecting fertility and seed set. However, effects of heat stress on reproductive structures and fertility in barley have not been extensively investigated. In this study we examined three commercial European spring barley varieties under high temperature conditions to investigate the effects on floret development. Using a combination of fertility assays, X-ray micro computed tomography, 3-dimensional modelling, cytology and immunolabelling, we observed that male reproductive organs are severely impacted by increased temperature, while the female reproductive organs are less susceptible. Importantly, the timing of stress relative to reproductive development had a significant impact on fertility in a cultivar-dependent manner, this was most significant at pollen mitosis stage with fertility ranged from 31.6-56.0% depending on cultivar. This work provides insight into how heat stress, when applied during male pollen mother cell meiosis and pollen mitosis, affects barley fertility and seed set, and also describes complementary invasive and non-invasive techniques to investigate floret development. This information will be used to identify and study barley cultivars that are less susceptible to heat stress at specific stages of floral development.

Scalable Biomimetic Syntheses of Paeciloketal B, 1-epi-Paeciloketal B, and Bysspectin A.

The first total synthesis of the benzannulated 5,5-spiroketal natural products paeciloketal B and 1-epi-paeciloketal B has been achieved in 10 linear steps employing a biomimetic spiroketalization. This approach also furnished the related natural product bysspectin A from the same putative biosynthetic precursor as the paeciloketals. Alternatively, bysspectin A could be accessed in only six steps using an improved route. This scalable and efficient synthesis affords insight into the biosynthesis of these natural products in nature.

Evolution and diversity of the angiosperm anther: trends in function and development.

KEY MESSAGE: Anther development and dehiscence is considered from an evolutionary perspective to identify drivers for differentiation, functional conservation and to identify key questions for future male reproduction research. Development of viable pollen and its timely release from the anther are essential for fertilisation of angiosperm flowers. The formation and subsequent dehiscence of the anther are under tight regulatory control, and these processes are remarkably conserved throughout the diverse families of the angiosperm clade. Anther development is a complex process, which requires timely formation and communication between the multiple somatic anther cell layers (the epidermis, endothecium, middle layer and tapetum) and the developing pollen. These layers go through regulated development and selective degeneration to facilitate the formation and ultimate release of the pollen grains. Insight into the evolution and divergence of anther development and dehiscence, especially between monocots and dicots, is driving greater understanding of the male reproductive process and increased, resilient crop yields. This review focuses on anther structure from an evolutionary perspective by highlighting their diversity across plant species. We summarise new findings that illustrate the complexities of anther development and evaluate how they challenge established models of anther form and function, and how they may help to deliver future sustainable crop yields.

The potential of resilient carbon dynamics for stabilizing crop reproductive development and productivity during heat stress.

Impaired carbon metabolism and reproductive development constrain crop productivity during heat stress. Reproductive development is energy intensive, and its requirement for respiratory substrates rises as associated metabolism increases with temperature. Understanding how these processes are integrated and the extent to which they contribute to the maintenance of yield during and following periods of elevated temperatures is important for developing climate-resilient crops. Recent studies are beginning to demonstrate links between processes underlying carbon dynamics and reproduction during heat stress, consequently a summation of research that has been reported thus far and an evaluation of purported associations are needed to guide and stimulate future research. To this end, we review recent studies relating to source-sink dynamics, non-foliar photosynthesis and net carbon gain as pivotal in understanding how to improve reproductive development and crop productivity during heat stress. Rapid and precise phenotyping during narrow phenological windows will be important for understanding mechanisms underlying these processes, thus we discuss the development of relevant high-throughput phenotyping approaches that will allow for more informed decision-making regarding future crop improvement.

The Synthesis and Bioactivity of the Marine Macrolide Callyspongiolide.

Callyspongiolide, a macrolide natural product with a conjugated diene-ynic side chain, has garnered significant attention from the synthetic community since its isolation from a sea sponge in 2013. Herein, the approaches that have been applied to this bioactive natural product to date are reviewed. These synthetic endeavors have established the absolute stereochemistry of this molecule and allowed further investigation into its promising caspase-independent bioactivity, while also contributing to the wider field of macrolide synthesis.

Molecules derived from the extremes of life: a decade later.

Covering: Early 2008 until the end of 2019Microorganisms which survive (extreme-tolerant) or even prefer (extremophilic) living at the limits of pH, temperature, salinity and pressure found on earth have proven to be a rich source of novel structures. In this update we summarise the wide variety of new molecules which have been isolated from extremophilic and extreme-tolerant microorganisms since our original 2009 review, highlighting the range of bioactivities these molecules have been reported to possess.

Increased expression of the MALE STERILITY1 transcription factor gene results in temperature-sensitive male sterility in barley.

Understanding the control of fertility is critical for crop yield and breeding; this is particularly important for hybrid breeding to capitalize upon the resultant hybrid vigour. Different hybrid breeding systems have been adopted; however, these are challenging and crop specific. Mutants with environmentally reversible fertility offer valuable opportunities for hybrid breeding. The barley HvMS1 gene encodes a PHD-finger transcription factor that is expressed in the anther tapetum, which is essential for pollen development and causes complete male sterility when overexpressed in barley. This male sterility is due at least in part to indehiscent anthers resulting from incomplete tapetum degeneration, failure of anther opening, and sticky pollen under normal growth conditions (15 °C). However, dehiscence and fertility are restored when plants are grown at temperatures >20 °C, or when transferred to >20 °C during flowering prior to pollen mitosis I, with transfer at later stages unable to rescue fertility in vivo. As far as we are aware, this is the first report of thermosensitive male sterility in barley. This offers opportunities to understand the impact of temperature on pollen development and potential applications for environmentally switchable hybrid breeding systems; it also provides a 'female' male-sterile breeding tool that does not need emasculation to facilitate backcrossing.

Rapid temperature responses of photosystem II efficiency forecast genotypic variation in rice vegetative heat tolerance.

A key target for the improvement of Oryza sativa (rice) is the development of heat-tolerant varieties. This necessitates the development of high-throughput methodologies for the screening of heat tolerance. Progress has been made to this end via visual scoring and chlorophyll fluorescence; however, these approaches demand large infrastructural investments to expose large populations of adult plants to heat stress. To address this bottleneck, we investigated the response of the maximum quantum efficiency of photosystem II (PSII) to rapidly increasing temperatures in excised leaf segments of juvenile rice plants. Segmented models explained the majority of the observed variation in response. Coefficients from these models, i.e. critical temperature (Tcrit ) and the initial response (m1 ), were evaluated for their usability for forecasting adult heat tolerance, measured as the vegetative heat tolerance of adult rice plants through visual (stay-green) and chlorophyll fluorescence (ɸPSII) approaches. We detected substantial variation in heat tolerance of a randomly selected set of indica rice varieties. Both Tcrit and m1 were associated with measured heat tolerance in adult plants, highlighting their usability as high-throughput proxies. Variation in heat tolerance was associated with daytime respiration but not with photosynthetic capacity, highlighting a role for the non-photorespiratory release of CO2 in heat tolerance. To date, this represents the first published instance of genetic variation in these key gas-exchange traits being quantified in response to heat stress in a diverse set of rice accessions. These results outline an efficient strategy for screening heat tolerance and accentuate the need to focus on reduced rates of respiration to improve heat tolerance in rice.

Ex vivo modeling of the airflow dynamics and two-and three-dimensional biomechanical effects of suture placements for prosthetic laryngoplasty in horses.

OBJECTIVE: To identify the degree of left arytenoid cartilage (LAC) abduction that allows laryngeal airflow similar to that in galloping horses, assess 2-D and 3-D biomechanical effects of prosthetic laryngoplasty on LAC movement and airflow, and determine the influence of suture position through the muscular process of the arytenoid cartilage (MPA) on these variables. SAMPLE: 7 equine cadaver larynges. PROCEDURES: With the right arytenoid cartilage maximally abducted and inspiratory airflow simulated by vacuum, laryngeal airflow and translaryngeal pressure and impedance were measured at 12 incremental LAC abduction forces (0% to 100% [maximum abduction]) applied through laryngoplasty sutures passed caudocranially or mediolaterally through the left MPA. Cross-sectional area of the rima glottis and left-to-right angle quotient were determined from photographs at each abduction force; CT images were obtained at alternate forces. Arytenoid and cricoid cartilage markers allowed calculation of LAC roll, pitch, and yaw through use of Euler angles on 3-D reconstructed CT images. RESULTS: Translaryngeal pressure and impedance decreased, and airflow increased rapidly at low abduction forces, then slowed until a plateau was reached at approximately 50% of maximum abduction force. The greatest LAC motion was rocking (pitch). Suture position through the left MPA did not significantly affect airflow data. Approximately 50% of maximum abduction force, corresponding to a left arytenoid angle of approximately 30° and left-to-right angle quotient of 0.79 to 0.84, allowed airflow of approximately 61 ± 6.5 L/s. CONCLUSIONS AND CLINICAL RELEVANCE: Ex vivo modeling results suggested little benefit to LAC abduction forces > 50%, which allowed airflow similar to that reported elsewhere for galloping horses.

MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis.

Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen-stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP-oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

Auxin export from proximal fruits drives arrest in temporally competent inflorescences.

A well-defined set of regulatory pathways control entry into the reproductive phase in flowering plants, but little is known about the mechanistic control of the end-of-flowering despite this being a critical process for optimization of fruit and seed production. Complete fruit removal, or lack of fertile fruit-set, prevents timely inflorescence arrest in Arabidopsis, leading to a previous proposal that a cumulative fruit/seed-derived signal causes simultaneous 'global proliferative arrest'. Recent studies have suggested that inflorescence arrest involves gene expression changes in the inflorescence meristem that are, at least in part, controlled by the FRUITFULL-APETALA2 pathway; however, there is limited understanding of how this process is coordinated at the whole-plant level. Here, we provide a framework for the communication previously inferred in the global proliferative arrest model. We show that the end-of-flowering in Arabidopsis is not 'global' and does not occur synchronously between branches, but rather that the arrest of each inflorescence is a local process, driven by auxin export from fruit proximal to the inflorescence apex. Furthermore, we show that inflorescences are competent for arrest only once they reach a certain developmental age. Understanding the regulation of inflorescence arrest will be of major importance to extending and maximizing crop yields.